Evaluation of Antimalarial Potential of Extracts from Alstonia boonei and Carica papaya in Plasmodium berghei-Infected Mice

Extracts of Alstonia boonei and Carica papaya are used in herbal medicine for the treatment of malaria. This work investigated the phytochemical, antioxidant, and antimalarial effects of hydromethanolic extracts of Alstonia boonei and Carica papaya. A four-day chemosuppressive test was conducted to assess the ability of the extracts to prevent establishment of infection. Three doses of the extracts were administered—100, 200, and 400 mg/kg bw—prior to Plasmodium berghei challenge. Change in body weight, parasitemia, packed cell volume (PCV), and mean survival time was determined. A three-day curative test was also carried out on Plasmodium berghei-infected mice to determine the effects of the plant extracts (200 mg/kg bw) on parasitemia and biochemical indices of liver and kidney functions, lipid metabolism, and oxidative stress. The study revealed that the extracts possessed phenolic compounds (34.13 ± 1.90 mg GAE/g for Alstonia boonei and 27.99 ± 1.46 mg GAE/g for Carica papaya) and flavonoids (19.47 ± 1.89 mg QE/g for Alstonia boonei and 18.24 ± 1.36 mg QE/g for Carica papaya). In vitro antioxidant activity measured as total antioxidant power, total reducing power, and DPPH radical scavenging activity showed that the extracts possessed higher antioxidant activity than the reference compounds. The outcome of the chemosuppressive test revealed that whereas Plasmodium berghei-infected mice had high parasitemia, decreased mean survival time, exhibited loss of weight, and had low PCV, treatment with the extracts reversed the effects in a concentration-dependent manner. Similarly, the curative test revealed that the extracts significantly suppressed parasitemia compared with the malaria negative control group. This was mirrored by reversal of indices of hepatic toxicity (AST, ALT, and ALP levels), nephropathy (urea and creatinine levels), oxidative stress (SOD, CAT, GPx, GSH, and lipid peroxides), and dyslipidemia (TC, HDL, and TG levels and HMG-CoA reductase activity) in infected but treated mice compared with negative control. Put together, the results of this study demonstrate that the extracts of Alstonia boonei and Carica papaya possess antimalarial properties and are able to ameliorate metabolic dysregulations that characterize Plasmodium berghei infection. The phytoconstituents in these extracts are believed to be responsible for the pharmacological activity reported in this study.


Introduction
Malaria still remains a threat to life especially in children below the age of 5 in sub-Saharan Africa. According to the latest world malaria report, over 400,000 deaths were reported from over 200 million infections in 2019. e report also shows that the gains in the fight against malaria seem to have plateaued in recent times [1]. is is partly due to resistance to current frontline antimalarial drugs involving artemisininbased combination therapy and the fact that the recently licensed malaria vaccine exhibits limited efficacy. e emergence of SARS-CoV-2 causing COVID-19 infections has further aggravated the scourge of malaria, limiting access to malaria health care services in the WHO African region due to travel restrictions. e fact that people with febrile illness are asked to stay at home as a preventive measure against the spread of SARS-CoV-2 also limits access to malaria treatment and could lead to a rise in malaria deaths in the nearest future if not properly managed. In the light of this, there is needed to look for potent alternatives of treating malaria in sub-Saharan Africa where the global burden of the disease is highest.
Medicinal plants have been used for a very long time to treat diseases, and they continue to be a pool for the discovery of drug leads [2]. In fact, current frontline antimalarial drugs are either derived directly from plants or are synthetically produced from a plant-derived chemical compound as template. For example, artemisinin was isolated from Artemisia annua [3], while quinine was isolated from Cinchona officinalis [4]. ese two compounds form the basis for artemisininbased combination therapy and the synthesis of chloroquine (or hydroxychloroquine), respectively. According to a recent review of plant-derived drugs and their contribution to the global disease pandemic, it is believed that the future of drug discovery lies in plant-derived natural products [5].
In Nigeria, folklore use of medicinal plants is a common practice due to accessibility, cultural acceptability, and relative affordability. However, this is usually at the risk of safety as most of the traditional remedies are without scientifically proven efficacy. Besides, the dosage of administration most of the time is arbitrary, increasing the risk of side effects and toxicity. One common practice among herbalists in Nigeria is the fact that these traditional remedies are derived from extraction using hydrophilic solvents such as water and ethanol. Some of the most common plants from which extracts are used as traditional remedies for diseases include Alstonia boonei, Carica papaya, Vitex doniana, Tapinanthus dodoneifolius, Senna alata, and Terminalia catappa. It is therefore expedient to study these fractions scientifically toward discovery of drug leads.
Alstonia boonei is a deciduous tree belonging to the family Apocynaceae. It can grow as high as 40 m and up to 27 m without branches [6]. Extracts from the plant have demonstrated antimalarial activities and management of anemia, which is characteristic of malaria infection. Its antimalarial activities have been examined using animal models both singly or in combination with other plants. For instance, Idowu et al. [7] showed that combination of Alstonia boonei with Picralima nitida, and Gongronema latifolium, had antiplasmodial activity with no evidence of toxicity to the liver or kidneys. A more recent study by Omoya and Oyebola [8], revealed that the leaves of Alstonia boonei had higher chemosuppressive activity than the stem extract. Extracts of Alstonia boonei have been reported to be rich in phytochemicals, which could target a plethora of plasmodium metabolic pathways.
One of such reports showed that a compound isolated from A. boonei inhibits the activity of both lactate dehydrogenase and plasmepsin II in malaria parasites [9]. Stem bark infusion of the plant has shown anticancer properties against colon cancer cell line [10] as well as antiulcerative properties [11]. Aqueous extracts of the leaf of this plant has also demonstrated anti-inflammatory and antioxidant activities in experimental rats [12].
Carica papaya is a plant commonly referred to as papaya or pawpaw. e fruit of the plant is eaten globally, while the leaves are used in some parts of the world to treat human diseases such as malaria, typhoid, piles, and diabetes. Literature search reveals scientific evidence for the antimalarial activity of C. papaya extracts when administered singly or in combination with other plant extract or with approved drugs [13][14][15][16]. C. papaya is rich in bioactive phytochemicals of diverse families. Glycosides of flavanols and caffeoyl derivatives obtained from the decoction of C. papaya leaves have shown synergistic potency with artesunate against P. falciparum and P. berghei, preventing parasite recrudescence [17].
However, despite the above reports, there are no available data on the antiplasmodial effect of aqueous and ethanolic extracts of the leaf of these plants to justify their use in traditional medicine for the treatment of malaria. erefore, this study seeks to provide scientific evidence for the use of aqueous and ethanolic extracts from the leaves of these plants locally in traditional medicine. Compounds identified from the phytochemical screen can be investigated further for the discovery of potent drug leads against malaria.

Total
Phenolics. e total phenolic of the extracts was determined using the Folin-Ciocalteu assay method of Singleton and Rossi [20]. e total phenolic content of the plants was then calculated and expressed as mg gallic acid equivalent (GAE)/g extract.

Total Flavonoids.
Total flavonoid content was determined using the aluminum chloride colorimetric method by Zhilen as described by Arogba [21]. e concentrations were expressed as mg quercetin equivalent (QUE)/g extract.

Total Antioxidant Power of Extracts.
e total antioxidant power of the extracts was evaluated according to the method described by Sharifia et al. [22]. Briefly, concentrations ranging from 25 to 400 µg/ml of the extracts were prepared in DMSO. 100 µ l of each concentration of the extracts was mixed with 1 ml of the working reagent (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). e reaction mixture was incubated at 95°C for 90 min and then cooled, and absorbance was taken at 695 nm against a blank.
Vitamin C was used as standard. e EC 50 (effective concentration) of the extracts was extrapolated from a plot of absorbance versus concentrations of each extract. e concentration that gave 0.5 absorbance was taken as the EC 50 .

Reducing Power of Extracts.
e reducing power assay was performed according to the method described by Yen et al. [23]. Plant extract with concentrations ranging from 25 to 400 µg/ml was mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of potassium ferricyanide (1% w/ v). e mixture was incubated at 50°C for 20 min. en, 2.5 ml of 1% trichloroacetic acid was added to the mixture to stop the reaction followed by centrifugation at 3000 rpm for 10 min. e supernatant (2.5 mL) was mixed with distilled water (2.5 mL) and 0.1% FeCl 3 (0.5 mL), and then absorbance was measured at 700 nm against a blank.

DPPH Radical Scavenging Property of Extracts.
e DPPH radical scavenging activity was evaluated according to the method of Blois [24]. IC 50 , which is the concentration at which 50% of the DPPH radical is scavenged, was determined from the curve of percentage inhibition plotted against various concentrations of the extracts. e results obtained were compared with the IC 50 of quercetin.

Experimental Animals and Malaria Parasite.
Male albino mice obtained from the animal house of Ahmadu Bello University, Zaria, Nigeria, were used for this study. e mice were kept in metal cages and allowed to acclimatize for the period of 14 days before been infected with the malaria parasite. Animals were fed commercial rat chow and water ad libitum throughout the period of the experiments. Animal sacrifice was humanely carried out according to acceptable international standards.
Plasmodium berghei NK65 was a gift from the Department of Parasitology, National Institute for Medical Research, Yaba, Lagos, Nigeria. e parasite was maintained in a mice host by serial passage of infected mouse to uninfected naïve mouse. e level of parasitemia was monitored by microscopic examination of blood smears.

Acute Toxicity Test.
e acute toxicity test in both plants was carried out according to OECD no. 425 guideline as described by Habte et al. [25,26]. Male mice were fasted overnight and administered with a single oral dose of 2000 mg/kg body weight of hydroethanolic extracts of Alstonia boonei and Carica papaya. e mice were fasted for additional two hours before given food and water. e mice were thereafter observed for 24 hours. e mice were further observed for 14 days for signs of toxicity such as convulsion, aggression, loss of appetite, hair erection, and muscle tremor.

Induction and Monitoring of Parasitemia.
Chloroquine-sensitive Plasmodium berghei NK65 strain was maintained in mice by passage of blood from infected to healthy mouse once every 4-5 days. Induction of mouse was done by intraperitoneal injection of 200 µl of blood (20-30% parasitemia) from an infected mouse (blood collected via cardiac puncture). Parasitemia was monitored by standard methods; thin blood smears were made on glass slides, fixed using ethanol, and stained using Giemsa stain, and parasitemia was counted using a microscope and was calculated as a percentage of infected red blood cells (RBCs) relative to the total number of cells in a microscopic field at ×100 magnification as given below:

Animal Sacrifice and Collection of Blood Samples.
At the end of the experiments, mice were anesthetized using chloroform in a glass jar and blood was collected via cardiac puncture. e blood was allowed to stand on the bench for 1 h to clot followed by centrifugation at 5,000 rpm for 10 min to separate serum.

Determination of Packed Cell Volume (PCV).
Packed cell volume (PCV) was determined using the capillary method. Tail blood was collected into a heparinized hematocrit tubes. e tubes were sealed with crystal seal and thereafter centrifuged for about 10,000 rpm for 5 minutes. e volume of cells was calculated according to the following formula: 2.6. Biochemical Assays. Diagnostic kits were used for the analysis of serum indices for kidney function (urea, creatinine), liver function (AST, ALT, ALP), lipid profile (HDL, TG, total cholesterol), HMG-CoA reductase, and oxidative stress markers (lipid peroxidation, catalase, superoxide dismutase, glutathione peroxidase). Assays were performed according diagnostic kits' manufacturer's protocols.

Statistical Analysis.
InStat GraphPad software was used for analysis of variance (ANOVA) to ascertain significant differences between means. Differences were considered statistically significant at P < 0.05.

Results and Discussion
Medicinal plants have been used for decades in the treatment of diseases, and they continue to be a pool for the discovery of drug leads [2]. e present study reports the antimalarial activity of Alstonia boonei and Carica papaya in P. bergheiinfected mice. is study showed that the hydroethanolic extracts of Alstonia boonei and Carica papaya contain alkaloids, flavonoids, phenolics, tannins, saponins, anthraquinones, and terpenoids (Table 1). Alkaloids, flavonoids, and tannins are known for their antioxidant, antimalarial, antimicrobial, anticancer, and anti-inflammatory activities [27,28]. e extracts had total phenolics (34.13 ± 1.90 mg GAE/g for Alstonia boonei and 27.99 ± 1.46 mg GAE/g for Carica papaya) and total flavonoid (19.47 ± 1.89 mg QE/g for Alstonia boonei and 18.24 ± 1.36 mg QE/g for Carica papaya). Phenolics have been reported to possess antioxidant, antimalarial, antimutagenic, anticancer, and anti-inflammatory capacities. e hydroxyl group substituent of aromatic benzene rings are responsible for this biological activity due to their capacity to eliminate or absorb free radicals and to chelate reactive oxygen species molecules. e potency of phenolics is proportional to the number of hydroxyl (OH) groups present in their aromatic rings [29]. It is believed that flavonoids exert their antimalarial activity by inhibiting fatty acid biosynthesis (FAS II) in the parasite [30,31]. Some flavonoids have also been reported to inhibit the influx of L-glutamine and myoinositol into infected erythrocytes [32].
Over the years, there have been reports validating the fact that plants do possess antioxidant activity though in varying degrees. In this study, it was observed (Figure 1) that the hydroethanolic extracts of Alstonia boonei and Carica papaya had substantial in vitro antioxidant activity almost comparable to ascorbic acid. is is not unconnected with the phytochemicals present in the extracts. e DPPH scavenging activity and TAC reported in this study also gave credence to the antioxidant activity of the extracts. e extracts of Alstonia boonei and Carica papaya did not cause any death within the 24 hours of acute toxicity test. Furthermore, observation of the mice for 14 days did not reveal any behavioral changes characteristic of toxicity. is shows that the extract may be safe for in vivo administration up to 2000 mg/kg. Our result of acute toxicity test is similar to the results of the study by Enechi et al., which showed that Alstonia boonei was tolerated up to a dose of 5000 mg/kg [33]. Similarly, Solikhah et al. reported zero death for mice administered the leaf extract of Carica papaya up to a dose of 3000 mg/kg [34]. Figures 2 and 3 show the chemosuppressive and curative antimalarial effects of the hydroethanolic extracts of Alstonia boonei and Carica papaya in Plasmodium berghei-infected mice. e extracts had a significant chemosuppressive and curative antimalarial effect on the infected mice when compared with the untreated group. is is believed to be as a result of some of the bioactive compounds such as flavonoids, phenolics, alkaloids, and anthraquinones present in the extracts [27][28][29]. e extracts reduced parasitemia and increased mean survival time in the treated group. It was also observed in this study (Figure 4) that though the extracts could not support increase in body weight and packed cell volume (PCV) while exerting their antimalarial effect, respectively, they were able to reduce the decreasing effect of Malaria is known to cause liver and kidney damage especially is severe cases. In fact, it is the first parasitic infection to be clearly associated with glomerular diseases in the tropical region [24]. Hepatic dysfunction and jaundice are common features of severe malaria [35][36][37]. e extracts decreased serum AST, ALT, and ALP in the treated groups when compared with the untreated group ( Figure 5); this indicates hepatoprotection. e increase in the ALP in the untreated group may be due to compromised cell membranes integrity. Similarly, the increase in the AST and ALT levels could be due to leakage from the hepatocytes due to damage by the parasite [38,39]. Severe malaria can lead to glomeruli disease condition as well as tubules and interstitial region disorders. It was observed in this study that the   is is seen in the decrease of urea and creatinine (biomarkers for kidney function) in the treated groups compared with the negative untreated group (Figure 6). An alternative explanation for disturbances in urea and creatinine concentrations could be due to sequestration of the parasite into the renal microvasculature bed, which may lead to ischemia [40]. Liver and kidney diseases associated with malaria are basically as a result of erythrocyte abnormalities.
Parasitized red cells tend to adhere to healthy erythrocytes, blood platelets, and capillary endothelium, and this in turn leads to formation of rosettes and clumps, which impair microcirculation [41].
It has been hypothesized that malaria parasites use cholesterol and phospholipids from its host, resulting in a decrease of serum HDL-cholesterol [42].
is and other factors are responsible for dyslipidemia in Plasmodiuminfected animals. e result from this study showed an increase in HDL-cholesterol of the groups treated with the extracts, while a decrease in HDL-cholesterol was observed in the untreated groups (Figure 7). e fast multiplication of intrahepatic Plasmodium necessitates a high demand of lipid for organelle biogenesis [43]. In a study where microarraybased approach to profile hepatocyte response to Plasmodium infection was utilized, the results revealed upregulation of genes coding for sterol synthesis and lipid metabolism.
is evidently might be responsible for the increased activity of HMG-CoA reductase activity of the pathway of cholesterol synthesis. Previous studies also reported increased concentrations in TG and VLDL and simultaneously a decrease in HDL and LDL in malarial infection condition [43]. e increase in the HDL and decrease in TG reported in the study in the treated groups could be indicative of the therapeutic potential of the extracts. e extracts caused increased in antioxidant enzymes (SOD, catalase, GPX, and GSH) activity while decreasing lipid peroxides concentration in the treated groups; this is in contrast with the untreated group where decrease in antioxidant enzymes activity and increase in lipid peroxides were observed (Figure 8). is result correlates with what was observed in the in vitro antioxidant assay where the extracts were seen to possess substantial antioxidant activity. e increased enzyme activity is an indication of integrity or intactness of the enzyme, while the decreased activity in      Evidence-Based Complementary and Alternative Medicine negative control group may be indicative of compromised enzyme integrity by the increased production of free radicals. e lipid peroxides level reduced while GSH levels increased significantly across the treatment groups. is result complements the ability of the extracts to increase the activity of antioxidant enzymes in parasitized mice. e decrease in peroxides and increase in GSH may be an indication of recovery from the metabolic anomalies elicited by Plasmodium infection.
Put together, phytochemicals present in hydroethanolic extracts of Alstonia boonei and Carica papaya demonstrated antimalarial activity in Plasmodium berghei-infected mice. e extracts reversed hepatic toxicity, nephropathy, oxidative stress, and dyslipidemia in infected mice.

Conclusions
e hydroethanolic extracts of Alstonia boonei and Carica papaya possess bioactive compounds responsible for the antioxidant and antimalarial activity reported in this study. Further studies should be directed toward identifying and characterizing these bioactive active compounds for proper utilization of their potentials in disease management.

Data Availability
All data are included in the article.

Conflicts of Interest
e authors declare no conflicts of interest.

Authors' Contributions
FOA contributed to conceptualization, funding acquisition, investigation, writing, and supervision. FMI prepared the draft and carried out study investigation; CON wrote and edited the paper and performed software analysis; HFH edited the paper, performed software analysis, obtained funding, and proposed methodology; SA edited the paper, performed software analysis, obtained funding, and performed experiments; and GEB obtained funding and resources and contributed to writing. All the authors have read and agreed to the published version of the manuscript.